Legionella pneumophila, the bacterium that causes Legionnaires’ disease, secretes a set of protein kinases that mimic human enzymes, hijacking host cells to create a safe replication chamber. Researchers will use mass spectrometry and high-throughput enzyme assays to map exactly how these bacterial kinases interact with human proteins and to screen for compounds that block them. This matters because Legionnaires’ disease cases are rising—2021 saw 2.4 cases per 100,000 people in Europe, a 38% increase in travel-associated infections—and multi-drug resistant bacterial strains are a growing global health emergency. Currently, almost nothing is known about the specific targets or regulation of these bacterial kinases, which limits their use as drug targets. If successful, this work could establish a new class of antimicrobial targets: anti-virulence drugs that disarm the bacterium without killing it, potentially reducing selective pressure for resistance. The analytical pipeline developed here could also be adapted to study similar uncharacterised kinases from other pathogenic bacteria, broadening the impact beyond Legionella alone.
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Context Life has evolved an abundance of protein-based molecular machines to detect and coordinate responses to environmental stimuli. This biological process is referred to as ‘cell signalling’ and is controlled by a series of functionally divergent and tightly regulated enzymes that introduce chemical modifications on to target proteins. One such class of enzymes are the protein kinases, which catalyse the reversible modification of protein substrates with a phosphate group (phosphorylation). Protein kinases (STKs) regulate virtually every aspect of life and are found ubiquitously within the genomes of eukaryotes and prokaryotes. Challenge While protein kinases are essential for normal cellular signalling, aberrant protein kinase activity and deregulation of kinase-dependent signalling pathways perturbs normal cellular functions and is frequently associated with a huge variety of human pathologies. As such, ~25 % of global drug development efforts are believed to target protein kinases. Pathogenic microbes produce an arsenal of secreted virulence effector proteins that have co-evolved to manipulate host signalling pathways and promote bacterial survival. Legionella pneumophila (Lp) is an opportunistically intracellular pathogenic bacteria and the causative agent of Legionnaires’ disease (LD), a serious form of pneumonia in humans. According to the European Centre for Disease Prevention and Control, 2021 marked the highest annual notification of LD (2.4 cases per 100,000 people) and a 38 % increase in travel-associated LD compared to the previous year. Lp secretes = 300 survival promoting effector proteins, including several eukaryote-like Ser/Thr protein kinases (eSTKs), which disrupt host cellular processes, contribute to pathogenicity, and produce a replication permissible intracellular compartment - the Legionella Containing Vacuole (LCV). Given marked structural similarities between human STKs and eSTKs, both of which use ATP as a co-factor, we predict that eSTKs can also be drugged using principles established for STKs. Furthermore, observed evolutionary deviations from the canonical kinase fold may allow development of selective compounds that differentiate between host and bacterial kinases. However, we currently understand very little about the function of Lp eSTKs, including substrate specificities, how they modulate cellular activities, or how they are themselves regulated. Answers to these fundamental questions are critical to establish eSTKs as suitable alternative molecular targets for antimicrobial therapies, which are urgently required to respond to the global health emergency posed by multi-drug resistant (MDR) bacteria, including isolates of Lp. Aims and Objectives We have designed an unbiased analytical Mass Spectrometry (MS) and biochemical pipeline to decode the regulatory host-pathogen interaction networks of a panel of established Lp-secreted eSTKs and to appraise their utility as targets for anti-virulence therapies. We will exploit proven technologies that have revolutionised eukaryotic protein kinase research and uncover the cellular activities of enigmatic bacterial eSTKs. We will employ a novel high-throughput enzyme assay platform to kinetically evaluate bacterial kinase activity and enable compound library screening for potential inhibitor molecules. Applications and Benefits Mapping of host-bacterial kinase signalling networks will provide crucial insights into the pathophysiological processes that underpin Lp infection and determine their suitability as novel molecular targets for the development of medicines to combat MDR bacteria. In this context, recent advances in metagenomics have revealed that unstudied kinases and kinase-like proteins of unknown function are prevalent throughout bacterial phyla. We anticipate that the optimised methodologies and analytical tools developed will also be readily adaptable to analyse unstudied kinase virulence factors from a variety of microorganisms.
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